The RNA picornaviruses have evolved wonderfully robust and effective mechanisms to express their proteins and override host defenses. Internal ribosomal entry sites (IRESes) allow these genomes to bypass normal translational requirements for 5 cap structures and lure the ribosomes into viral instead of cellular pathways. The captured ribosomes pass down a single, long ORF, creating polyproteins that are tandem linkages of all structural and enzymatic units necessary for virulent infection. Individual proteins are liberated in an elaborate proteolytic cascade that is a defining feature of this family. At least three viral-encoded enzymes are required for complete processing, none of which has an exact cellular analogue. This program focuses on the murine cardioviruses, encephalomyocarditis virus (EMCV) and Mengovirus. The goals are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family,and to exploit the unique features of cardioviruses to examine fundamental molecular questions about picornaviral translation, proteolytic processing, replication and morpho genesis. The special propensity of cardiovirus RNAs to facilitate translation in cell-free extracts, the remarkable avidity of the processing cascade, the availibility of antibodies, enzyme assays, cDNAs, and crystal structures make these isolates exceptionally useful experimental subjects for molecular dissection of the virus lifecycle. New findings support an unexpected nucleolar localization for several non-structural proteins including protein 2A, and their participation in the induction of new ribosomes, apparently modified for the preferential translation of viral IRESes. Host mRNA synthesis is also shutoff after nuclear targeting by viral protease 3C. Although picornavirus replication was previously assumed to be exclusively cytoplasmic, these novel findings help explain the ease with which cardioviruses establish themselves during infection, and with which they bring about the (nefarious) abrogation of nearly any type of cellular defense system. Additional data support an unusual cryptic cleavage in replication protein 2B, and point to an alternate processing cascade activated. during translational stress. The pathway is another example of flexibility in the cardioviral lifecycle and the subtle but dynamic mechanistic balance between viral translation and replication. The objectives set forth in this proposal build directly upon experimental foundations developed during the preceding years of the program. The specific alms are: (1) to probe the mechanism by which Mengoviral protein 2A localizes to nucleoli, incorporates into modified ribosomes, and causes the shutoff of host protein synthesis; (2) to examine the relationship between nuclear localization of viral proteins (3Cpro, 3Dpol and 3B), the shutoff of cellular mRNA transcription, and the upregulation of rRNA transcription during infection; (3) to determine the role of a conserved, cryptic 3Cpro cleavage site within viral protein 2B, and document its cleavage accessibility during infection: and (4) to explore Mengoviral Leader protein cleavage and phosphorylation as regulatory triggers for Leader/IRES interactions.